To gain further understanding of the mechanisms by which trans-acting factors interact to recognize transcriptional regulatory sequences, we have examined the ability of simian virus 40 (SV40) T-antigen and adenovirus E1A protein to stimulate the adenovirus E2 promoter. An E2-CAT plasmid was cotransfected into monkey kidney cells with plasmids encoding SV40 T-antigen or E1A. CAT activity and RNA is then assayed to estimate the rate of transcriptional activity directed from the E2 promoter. We have found that either SV40 T-antigen or E1A protein will stimulate the E2 promoter in cultured monkey kidney cells (CV-1 cells). Based on experiments in which concentrations of the E2-CAT plasmid or T or E1A-encoding plasmids were varied, it appears that the mechanism by which T and E1A activate the E2 promoter are different. Furthermore, T-antigen in COS-1 cells, which are derived from CV-1 cells by transformation with SV40 and constitutively express T-antigen, does not stimulate the E2-CAT plasmid. E1A, however, is able to stimulate this plasmid in COS-1 cells. This suggests that there are at least two different mechanisms for activation of the E2 promoter, and only the E1A-mediated mechanism is active in COS-1 cells. This also suggests that the different activities of SV40 T-antigen may be separable, since SV40 replication and late gene expression is observed in COS-1 cells. Using synthetic oligonucleotide promoter sequences, we are currently examining whether there are sequences on the E2 promoter which can distinguish between T-antigen and E1A-mediated stimulation. In addition, stereochemical interactions between proteins binding to individual upstream promoter sequences are under investigation.